1. Field of the Invention
This invention relates to nucleic acid encoding polypeptides capable of synthesizing compounds having anti-fungal activity, methods for their preparation, and methods for their use.
2. Description of Related Art
Polyketides are complex natural products that are produced by microorganisms such as fungi and mycelial bacteria. There are about 10,000 known polyketides, from which numerous pharmaceutical products in many therapeutic areas have been derived, including: adriamycin, epothilone, erythromycin, mevacor, rapamycin, tacrolimus, tetracycline, rapamycin, and many others. However, polyketides are made in very small amounts in microorganisms and are difficult to make or modify chemically. For this and other reasons, biosynthetic methods are preferred for production of therapeutically active polyketides. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; WO 97/02358; and WO 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146 and 6,410,301; Fu et al. (1994), Biochemistry 33:9321-26; McDaniel et al. (1993) Science 262: 1546-1550; Kao et al. (1994) Science, 265:509-12, and Rohr (1995) Angew. Chem. Int. Ed. Engl. 34: 881-88, each of which is incorporated herein by reference.
Biosynthesis of polyketides may be accomplished by heterologous expression of Type I or modular polyketide synthase enzymes (PKSs). Type I PKSs are large multifunctional protein complexes, the protein components of which are encoded by multiple open reading frames (ORF) of PKS gene clusters. Each ORF of a Type I PKS gene cluster can encode one, two, or more modules of ketosynthase activity. Each module activates and incorporates a two-carbon (ketide) unit into the polyketide backbone. Each module also contains multiple ketide-modifying enzymatic activities, or domains. In classical Type I PKSs, the number and order of modules, and the types of ketide-modifying domains within each module, determine the structure of the resulting product. Recently, variants of Type I PKSs have been found in which single modules may be used in an iterative fashion to add more than one two-carbon unit to the growing polyketide chain (see, for example, Müller (2004) Chem. Biol. 11(1):4-6). Polyketide synthesis may also involve the activity of nonribosomal peptide synthetases (NRPSs) to catalyze incorporation of an amino acid-derived building block into the polyketide, as well as post-synthesis modification, or tailoring enzymes. The modification enzymes modify the polyketide by oxidation or reduction, addition of carbohydrate groups or methyl groups, or other modifications.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker regions. These regions collectively can be considered to define boundaries of the various domains. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) from other PKSs by various available methodologies. Using this method, new polyketide synthases (which produce novel polyketides) can be produced. It will be recognized from the foregoing that genetic manipulation of PKS genes and heterologous expression of PKSs can be used for the efficient production of known polyketides, and for production of novel polyketides structurally related to, but distinct from, known polyketides (see references above, and Hutchinson (1998) Curr. Opin. Microbiol. 1:319-29; Carreras and Santi (1998) Curr. Opin. Biotech. 9:403-11; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference).
One valuable class of polyketides includes the ambruticins and their analogs (FIG. 1), produced by the myxobacterium Polyangium cellulosum (also known as Sorangium cellulosum) var. fulvum, deposited as ATCC 25532. Ambruticin S (also known as “Acid S” or “W7783”) was described in U.S. Pat. Nos. 3,651,216 and 3,804,948. Ambruticins VS1, VS3, and VS5 and a fermentative process for producing them using Sorangium cellulosum So ce10, deposited as DSM 5386, are disclosed in PCT publication WO91/00860. The ambruticins are anti-fungal agents. It is believed that the ambruticins kill fungi through interference with osmoregulation (Knauth and Reichenbach (2000) J. Antibiotics 53:1182-1190).
Given the promise of ambruticins in the treatment of fungal infections, there exists an unmet need for a production system that can provide large quantities of ambruticins. The present invention meets this need by providing the biosynthetic genes responsible for the production of ambruticins and providing for their expression in heterologous hosts.